Current Reviews in Musculoskeletal Medicine (2020) 13:264–280 https://doi.org/10.1007/s12178-020-09624-0

STEM CELLS IN ORTHOPAEDIC SURGERY (J DRAGOO AND K JONES, SECTION EDITORS)

Adipose-Derived Treatments and Available Formulations

Kyle N. Kunze1 & Robert A. Burnett1 & Joshua Wright-Chisem2 & Rachel M. Frank3 & Jorge Chahla1

Published online: 23 April 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract Purpose of Review The use of human adipose-derived mesenchymal stem cells (ADSCs) has gained attention due to its potential to expedite healing and the ease of harvesting; however, clinical evidence is limited, and questions concerning optimal method of delivery and long-term outcomes remain unanswered. Recent Findings Administration of ADSCs in animal models has been reported to aid in improved healing benefits with enhanced repair biomechanics, superior gross histological appearance of injury sites, and higher concentrations of growth factors associated with healing compared to controls. Recently, an increasing body of research has sought to examine the effects of ADSCs in humans. Summary Several available processing techniques and formulations for ADSCs exist with evidence to suggest benefits with the use of ADSCs, but the superiority of any one method is not clear. Evidence from the most recent clinical studies available demonstrates promising outcomes following treatment of select musculoskeletal pathologies with ADSCs despite reporting variability among ADSCs harvesting and processing; these include (1) healing benefits and pain improvement for rotator cuff and Achilles tendinopathies, (2) improvements in pain and function in those with knee and hip osteoarthritis, and (3) improved cartilage regeneration for osteochondral focal defects of the knee and talus. The limitation to most of this literature is the use of other therapeutic biologics in combination with ADSCs. Additionally, many studies lack control groups, making establishment of causation inappropriate. It is imperative to perform higher-quality studies using consistent, predictable control populations and to standardize formulations of ADSCs in these trials.

Keywords Adipose . Stem cells . ADSCs . Biologics . Formulations . Regenerative medicine

Introduction that have the ability to differentiate into several lineages, is one such adjunct with increasing interest as they can be utilized for The use of biologic adjuncts as both operative and non- clinical benefit by expediting physiological processes responsi- operative treatments for orthopedic pathologies has become of ble for healing. Several sources from adult progenitor cells have recent interest due to potential healing benefits. [1–5] been reported in the literature including marrow mesen- Administration of progenitor cells, defined as pluripotent cells chymal stem cells and peripheral blood stem cells; [6, 7]more recently, adipose-derived mesenchymal stem cells (ADSCs) This article is part of the Topical Collection on Stem Cells in Orthopaedic have been recognized as an alternative source of stromal cells Surgery thought to have equivalent differentiation capacity. A primary reason for increased interest in the use of ADSCs * Jorge Chahla for clinical applications manifests from the ease with which they [email protected] are harvested relative to other stromal cells, as subcutaneous stores in the infrapatellar fat pad and buttocks/flank allow for a 1 Department of Orthopaedic Surgery, Division of Sports Medicine, less invasive isolation process with a similar multi-lineage poten- Rush University Medical Center, Chicago, IL, USA tial. [8] Indeed, these minimally invasive harvesting techniques 2 Department of Orthopaedic Surgery, Hospital for Special Surgery, are associated with lower donor site morbidity and pain and New York, NY, USA fewer complications than the harvesting of other stromal cells, 3 Department of Orthopaedic Surgery, Division of Sports Medicine, and lipoaspirate has been demonstrated to result in higher University of Colorado School of Medicine, Boulder, CO, USA Curr Rev Musculoskelet Med (2020) 13:264–280 265 progenitor cell yields than aspirates [9]. Other pro- osteogenic-specific molecular pathways which utilize migra- posed benefits of ADSCs include greater availability, with re- tion, molecular adhesion, and differential signaling to increase ports of up to 10% nucleated cells from ADSCs versus 0.001– bone production. [22] Most theories accept the premise that 0.01% of bone marrow-derived mesenchymal stem cells, a low these cascades begin with a commitment step from the stem rate of complications, and high proliferative potential and rapid cell precursor into the osteogenic lineage through molecular expansion. [10–13] signals including transforming growth factor (TGF)-β,bone Despite increasing use, several limitations to ADSCs still morphogenic protein (BMP) family, fibroblast growth factor exist. While controlled laboratory studies involving the use of (FGF), and Wnt/β-Catenin proteins. [22] The cellular and ADSCs as an augment for the treatment of common orthope- molecular mechanisms that underlie osteogenic differentiation dic pathologies such as focal osteochondral defects and rotator via ADSCs are complex and out of scope of the current re- cuff tendinopathy have reported promising results in animal view; however, few major mechanisms and prevalent theories models, [14–17] few studies involving human subjects exist. of osteogenic differentiation are presented below. [18–21] Furthermore, most of these studies have inconsistent BMP has been implicated as one of the primary initiators of protocols and formulations of ADSCs, making it difficult to the signaling cascade for ADSC-mediated osteogenesis. compare results between studies. Additionally, it is still un- Studies have shown that BMP initiates the cascade via ligand clear if ADSCs act directly by repairing the tissue or through binding of serine/threonine kinase cell surface receptors. [23] signaling environmental molecules to aid the repair of the The next step involves these activated receptor kinases phos- tissue. [22] The current article presents a comprehensive re- phorylating transcription factor SMADs, which subsequently view of the mechanisms of action of ADSCs from the basic form a heterodimeric complex and promote gene expression science literature and available formulations of ADSCS. and osteogenic differentiation. [24] BMP-2 induces the phos- Additionally, this review discusses evidence for the safety phorylation of Smad 1/5/8 and the transactivation of a BMP/ and efficacy of ADSCs for orthopedic pathologies and injuries Smad-responsive construct (12xSBE-Oc-pGL3). [25] in both animal models as well as clinical trials with an empha- Furthermore, Ducy et al. [26] showed that BMPs were able to sis on clinical and functional outcomes as well as variability in increase the transcription of core-binding factor-1/Runt-related harvesting and processing protocols. Finally, future directions family 2(Cbfa1/Runx2), a molecule that is well known to be for the use of ADSCs in the context of the current evidence in essential for the commitment process of osteoblastic lineage. this review will be presented. The Notch pathway has also been found to be heavily implicat- ed in the role of osteogenic differentiation through interactions with BMP-2, which results in increased expression of Delta1/ Proposed Mechanisms of Action Jagged1-activated Notch. [25, 27] Interestingly, overexpression from the Basic Science Literature of the Notch intracellular domain (NICD) impairs osteogenesis. Notch has been demonstrated to oppose the effects of BMP-2 The multipotent nature of ADSCs allows for the potential to and Wnt-3a on alkaline phosphatase activity, which is normally differentiate into several different lineages including osteo- upregulated by these markers. Furthermore, Notch overexpres- genic, chondrogenic, adipogenic, and myogenic cell lines. sion decreases the transactivating effect of Wnt-3a, cytoplasmic Given the proposed beneficial effects of the use of ADSC in β-catenin levels, and Wnt-dependent gene expression. NICD bone, tendon and muscle healing, a better understanding of the overexpression prevents BMP-2 and Wnt biological effects by downstream effects induced by ADSC at the molecular level suppressing Wnt, but not BMP signaling. [25] This is a clini- is imperative to clarify how these outcomes are mediated. cally relevant consideration in appropriately culturing and ma- Furthermore, such clarification will help better understand nipulating cellular conditions to optimize osteogenic differenti- ADSCs as a biologic adjunct in general, the spectrum of their ation from ADSCs. use in repair of common musculoskeletal injuries, and poten- Grottkau et al. [28] harvested ADSCs from normal rats and tially aid in the optimization of ADSCs formulations. attempted to utilize BMP-2 enhanced ADSCs to restore criti- cal size cranial defects. There were three populations studied: Proposed Mechanisms of Osteogenesis one group with deficits filled with alginate gel alone, another with alginate gel and normal ADSCs, and finally alginate gel While the specific mechanisms of osteogenesis from ADSCs with BMP-2 transfected ADSCs. The study concluded that are complex and not completely understood, there are several alginate gel with BMP-2-enhanced ADSCs was necessary proposed mechanisms by which osteogenic differentiation is for critical size defect repair. thought to occur. Recent research has focused specifically on While molecular influences are essential for the differenti- signaling pathways and molecular biology. In particular, oste- ation of ADSCs, the mechanical environment can play a role ogenic differentiation is thought to proceed through the up- in osteoblastic differentiation. [29] Rath et al. demonstrated regulation of various growth factors and acceleration of that mechanical stimulation may function as an anabolic 266 Curr Rev Musculoskelet Med (2020) 13:264–280 stimulus for the osteogenic differentiation of mesenchymal govern this process. Indeed, it appears that function of the stem cells. [29] Yang et al. [30] demonstrated that mechanical JNK pathway has a large role in committing to osteogenic influences are also implicated in the differentiation of ADSCs. differentiation of ADSCs in vitro. [34] This group osteo-induced ADSCs for 48 hours by culturing Interestingly, it has also been demonstrated that ADSCs them in a medium containing α-MEM supplemented with have the potential to seed and integrate into porous metal 10% FBS, 0.1 μM dexamethasone, 10 mM glycerol phos- prosthetics after osteogenic induction. Lewallen and col- phate, and 50 mM L-ascorbic acid-2-phosphate. Following leagues [35] cultured human ADSCs on surgical-grade porous osteoinduction, the authors then placed them under uniaxial titanium disks as models for orthopedic implants and loads of various short or long duration patterns. This study osteogenically induced them. They found that ADSCs ad- showed that with increased duration of continuous mechanical hered well to the porous surface and grew into the porous stress, gene expression of BMP-2 and Runx2 were significant- microenvironment compared to controls. Together, this sug- ly increased. Furthermore, they concluded that ADSCs may gests that ADSCs may have a role in clinical cases where sense mechanical loading in a duration-dependent manner, enhanced fixation of surgical components is required. which affects osteogenic differentiation of ASDCs via the Namely, if there is significant bone loss or deformity, it is BMP-2 pathway. In addition to tensile loading, fluid flow possible that ADSCs are pre-programmed to express an oste- and shear stress have been shown to be implicated in osteo- oblastic phenotype to help restore these deficits. genic proliferation of ADSCs. [31] In fact, pulse fluid flow (PFF) has been shown to directly stimulate osteogenic differ- entiation via fluid shear in ADSCs. Proposed Mechanisms of Chondrogenesis Another proposed mechanism of osteogenic differentiation via ADSC precursors is resultant expression and production of There are at least four major mechanisms by which ADSCs the bone marker proteins including alkaline phosphatase, type may be manipulated towards chondrogenic differentiation and I collagen, osteopontin, and osteocalcin through the aforemen- may result in a clinically relevant cell source for regenerative tioned molecular pathways. These markers are involved in medicine: (1) response to specific growth factors, (2) utiliza- mineralized matrix production. In a study comparing in vitro tion of specific culture media, (3) induction by low oxygen differentiation potential between various mesenchymal stem tension, and (4) culture with novel biomaterial scaffolds. cell types, ADSCs induced the expression of alkaline phos- The first mechanism through which chondrogenic differen- phatase and upregulated Runx2 gene expression, both of tiation may be selected for is by subjecting ADSCs to a spe- which are highly implicated in regeneration and mineraliza- cific growth factor milieu. Similar to osteogenesis, the propen- tion. [32]D’Alimonte et al. [33]soughttocomparetheeffects sity for ADSCs to differentiate into chondrocytes is highly of human ADSCs and dental-pulp stem cells (DPSCs) and dependent on culture conditions. Most standard protocols uti- found that ADSCs conferred greater osteogenic differentiation lize growth factors from the TGF-β superfamily to promote as measured by higher levels of expression of various early chondrogenesis; [36] however, numerous protocols utilizing a osteogenic markers including Runx2 and alkaline phospha- wide variety of culture conditions exist, most of which require tase at days 3–14, in addition to extracellular matrix mineral- 3 to 4 weeks for complete differentiation. It is thought that the ization at days 14–21. Furthermore, the authors found that TGF-β superfamily of growth factors facilitates the differen- ADSCs demonstrated faster doubling time and colony- tiation of ADSCs through several signaling pathways includ- forming ability when compared to DPSCs. The authors con- ing extracellular signal-related kinase-1 and 2, SMAD, and c- cluded that ADSCs could be effective in regenerative ortho- Jun-N-terminal kinase. [37–39] Indeed, culture with TGF-β pedics given these properties. and BMP-6 has been shown to induce cartilage-specific pro- Culture formula and conditions may also play a role in teins including type II collagen. [39, 40] A recent study in- favoring osteogenic differentiation. Based on the fact that duced ADSCs with transforming growth factor beta-3 (TGF- mesenchymal precursor cells must undergo proliferation and B3) and bone morphogenetic protein-6 (BMP-6) and found differentiation and can be manipulated to do so by varying that over 2–4 weeks, chondrocytes were formed and were formula conditions, Gu et al. [34] sought to investigate the surrounded by type II collagen and aggrecan. [41] use of osteogenic formula induction on ADSCs. After induc- Interestingly, a total of 10 major chondrogenic genes were ing ADSCs, the authors found increased c-Jun N-terminal upregulated in this study. Ultimately, , immunohisto- kinase (JNK) activation at days 13–17, and this was associated chemistry, and gene expression profiles confirmed that the with extracellular matrix synthesis and increased calcium de- ADSCs were transformed into hyaline-like cartilage in cul- position, the two hallmarks of bone formation. The authors ture. The authors of this study proposed that ADSCs may have proposed that the MAP kinase pathway was therefore a po- a significant role in joint restoration given the current chal- tential mechanism in ADSC proliferation and differentiation lenges incurred with degenerative joint disease. Other growth and that extracellular receptor kinase (ERK) signaling helps to factors which have demonstrated a strong chondrogenic Curr Rev Musculoskelet Med (2020) 13:264–280 267 potential include BMP-4, sex determining region Y box 9 I, II, X, and keratin and chondroitin sulfate; (2) polymerase (SOX 9), and basic fibroblast growth factor. chain reaction; (3) Western blot analysis and ELISA; and (4) The second mechanism through which chondrogenic dif- RNA microarrays. Genomic analysis is capable of identifying ferentiation may be selected for is by ADSCs is through the the stages of chondrogenic differentiation as differential gene utilization of media which favors chondrogenesis. Three com- expression is indicative of specific stages within the monly used protocols are implicated in promoting the chondrogenic lineage. [46] Early in stage 1, SOX 4, BMP-2, chondrogenic differentiation of ADSCs by manipulating the and collagen I and VI are expressed; in stage 2, collagen XI, media to select for this lineage. Each protocol utilizes a com- SOX 9, and COMP are expressed; in stage 3, Homeobox 7, bination of a medium with various supplements: (1) Wnt, chondroadherin, and Indian hedgehog are expressed; Dulbecco’s modified Eagle’smedium(DMEM)with and as chondrogenesis approaches completion in stage 4, TGF-β3, albumin (1.25 μg/mL), dexamethasone (10-7 M), osteocalcin, fibromodulin, alkaline phosphatase, aggrecan, ascorbic acid (6.25 μg/mL), transferrin, and insulin PTHrP, and collagens type II, IX, and X are expressed. (6.25 μg/mL); [42] (2) DMEM + 1% fetal calf serum (FCS) Taken together, chondrogenic differentiation of ADSCs is with TGF-β1 (10 nh/mL), ascorbate-2-phosphate (50 nM), an intricate process highly dependent on growth factor milieu and insulin (6.25 μg/mL); [43] and (3) OriCell (Cyagen, and culture conditions; although common methods exist, there GUXMX-90041, Santa Clara, CA, USA) with TGF-β3, dexa- is no consensus as to which combination of conditions opti- methasone, ascorbic acid, ITS cell culture supplement, sodium mizes the yield of ADSCs. Chondrogenic differentiation may pyruvate, and proline. also be tracked by identifying differential gene expression at The third mechanism through which chondrogenic differ- various stages over the 3–4-week culture length. entiation may be selected for is through subjecting ADSCs to low oxygen tension. Creating a hypoxic environment in con- junction with specific culture conditions, studies have demon- strated that restricting oxygen during this process may en- Available Formulations hance the yield and selectivity of chondrogenesis. [39] Specifically, restricting ADSC culture conditions to 5% oxy- Bone marrow-derived MSCs (BM-MSCs) are traditionally gen has been demonstrated to be selective for chondrogenesis. harvested from cancellous bone within the iliac crest, proxi- However, there is currently no evidence to suggest that this mal tibia, proximal humerus, and calcaneal tuberosity. effect remains in vivo. [44] However, utilization of BM-MSC has recently come under The fourth mechanism by which ADSCs can be manipu- scrutiny. The harvesting of these cells is invasive and initial lated to favor the pathway of chondrogenesis is by promoting studies suggest that cell yield is lower than ADSCs. [49–54] growth by utilizing scaffolds and mechanical forces. [39]Itis Moreover, there is concern regarding the pluripotency of BM- proposed that mechanical forces take advantage of influencing MSC, with evidence suggesting an inverse relationship be- ADSC cell morphology through responses to extracellular tween differentiation potential and donor age. [55] For the stress. [45] This may be accomplished through applying pres- above-mentioned reasons, alternative sources of multipotent sure to cell cultures with dynamic stamps or centrifugal forces. stem cells, such as ADSCs, are of great interest to orthopedic Likewise, commercial systems exist which are designed to surgeons and patients alike (Table 1). take advantage of this mechanical force concept. The ADSCs are a particularly promising therapy given the low- FlexCell system (FlexCell Tension System FX-5000T, Dunn er morbidity profile associated with the harvesting procedure, Labortechnik GmbH, Asbach, Germany) depends on seeding often being completed as an in-office procedure. Additionally, ADSCs onto silicone membranes which are stretched in a initial in vitro studies boast greater yields in ADSC harvests static or cyclic manner by changing the pressure environment. compared to bone marrow harvests. [53] BM-MSCs consti- Three-dimensional scaffolds and gel materials may also be tute only 0.002% of total stromal cell populations. [56] used to overcome growth inhibition imparted by cell-cell con- ADSCs, however, are considered to be the highest yield of tact in vitro by mimicking the physiologic milieu or utilizing all tissue types, with nearly 2% of MSC in the stromal vascular chemotactic agents to direct migration. [46, 47] fraction. [57] ADSCs have been isolated from the abdominal As the process of chondrogenesis proceeds, numerous lab- oratory techniques are capable of monitoring the differentia- Table 1 Benefits of tion process, [48] and in some cases identify which stage the ADSC relative to • Higher cell yield ADSCs are currently in with respect to completion of traditional BM-MSC use • Less invasive harvesting the differentiation process. These methods utilize detection • Lower risk of donor site infection of genes or markers expressed by ADSCs that are indicative • More predictable cellular differentiation of the stages of chondrogenic differentiation. Most commonly, • Less donor site pain the following are used: (1) immunostaining for collagen types 268 Curr Rev Musculoskelet Med (2020) 13:264–280 area, buttocks, flank, thigh, as well as infrapatellar fat pad supernatant and resuspension of concentrated pellets. The li- (IFP). [49] The buttocks are a common location to harvest posuction aspirate can then be expanded under sterile labora- ADSCs due to high yield with similar differentiation potential. tory conditions with sequential media refeedings over the next [19] Dragoo et al. demonstrated that IFP is a convenient har- 7 days. [59] The cells are harvested via trypsinization and vest location for patients already undergoing knee arthroscopy pelleted via centrifugation. From here, the isolated ADSC with a mean yield of 4.86 ± 2.64 × 105 cells/g of SVF tissue, sample can now be considered a dynamic source of sufficient for an autologous source of stem cells. [10]ADSCs multipotent stem cells that can be utilized in many different isolated from the buttock/flank and IFP offer a stable popula- ways: (1) further passage (up to 4 passages before concern for tion with low levels of senescence that can differentiate senescence), (2) freezing for further use, or (3) osteogenic or in vitro into adipogenic, chondrogenic, myogenic, and osteo- chondrogenic differentiation in pellet culture or alginate beads genic cells in the presence of lineage-specific induction fac- (Pronova LVG USP). [59, 64, 65] tors. [9, 58] The sources of variability in different enzymatic isolation The harvesting methods for ADSCs are well described and techniques primarily involve the following steps during pro- continue to see advancements as new technologies become avail- cessing: (1) number of washing steps, (2) enzyme concentra- able. [59] In 1994, Coleman et al. described a technique for har- tions, (3) centrifugation parameters, (4) erythrocyte lysis vesting subcutaneous which is still utilized today. methods, and (5) culture conditions. [63, 66–68] Concerns [60] A small incision is made over the desired region of adipose over the high cost, as well as senescence and decline in tissue, typically in the buttocks or flank. A 3-mm, blunt-edged, 2- multipotency with enzymatic processing techniques, have en- hole cannula connected to a 10-mL syringe is used to suction fat couraged the development of other technologies that rely on manually with a plunger, advancing the cannula through the har- mechanical means of isolation. [63, 69] As discussed in the vest site while applying negative pressure to isolate the adipose future directions section, reducing variability in these proto- tissue in the syringe. [60] The cannula is replaced with a Luer- cols will be imperative to allow for homogenous comparisons Lock aperture and the aperture is removed prior to the device being between studies when considering the efficacy and safety as placed in the centrifuge. [60] This method can be performed via a well as outcomes for the use of ADSCs in clinical trials. “wet” or “dry” method. [61]The“wet” method utilizes an injec- Potential non-enzymatic solutions for separation of ADSCs tion in the donor site that contains 0.9% NaCl, epinephrine, and a from lipoaspirate include use of shear force, gravity separa- local anesthetic, whereas the “dry” method does not employ use of tion, centrifugal force, vibration energy, and pressure. A num- tumescent fluid. [61] Illouz and de Villers demonstrated that the ber of automated devices on the market claim to offer a repro- “wet” technique can facilitate fat aspiration via hydrodissection. ducible, viable SVF sample for injection into the diseased or [62] IFP is harvested arthroscopically with use of a shaver and injured tissue of interest (Table 2). curette. The adipose tissue is removed from the intra-articular Centrifugation is a mechanical technique that capitalizes on space through arthroscopic portal and placed into a reservoir for the varying molecular weights of the lipoaspirate contents to fat isolation. The benefit of this procedure is that it can often be separate the SVF from residual lipids and inflammatory markers. associated with arthroscopic knee surgery, thus avoiding a separate The AdiPrep® Adipose Transfer System (Adiprep, Harvest liposuction procedure. Technologies Corporation, Plymouth, Mass.) is a commercially Significant variety exists among protocols for ADSC iso- available system which isolates adipose tissues from lipoaspirate lation; however, the technique described below offers a fairly using a proprietary lipid barrier disc technology that aims to standard method of enzymatic preparation in the United achieve more effective layer isolation than standard gravity de- States. [59] Typically, the lipoaspirate is mixed with a colla- cantation methodologies. [10, 77] Dragoo et al. [10] described a genase mixture consisting of 0.1% (w/v) collagenase promising novel application of this technique using modern in- (Worthington Biochemical Products, Lakewood, NJ), 1% (v/ strumentation for isolating ADSCs from arthroscopically har- v) bovine serum albumin (fraction V) (Atlas Biologicals, Fort vested IFP and surrounding synovium. The IFP-derived sample Collins, CO), and 2 mM calcium chloride in phosphate- is collected into a lipoaspirate filtration system (AquaVage, M.D. buffered saline (PBS, Gibco, Fisher Scientific). [59] Resource, Livermore, Calif.) followed by fat fractionization with Alternative enzymes used for fat separation include trypsin a syringe emulsification technique and finally concentrated with and dispase. [63] After a period of incubation, the blood layer the AdiPrep® Adipose Transfer System. They report successful is aspirated off leaving a layer of fat, which is then washed SVF isolation with mean yield of 4.86 ± 2.64 × 105 cells/g of several times with D-PBS (1:1 volume) for further isolation. tissue and a mean viability of 69.03% ± 10.75%, achieving the Occasionally, an erythrocyte lysis step is included to extract standard cutoffs for therapeutic benefits of treatment. This meth- erythrocyte contamination and to decrease the amount of he- od provides physicians with a simple means of obtaining autol- matopoietic progenitor cells. The fat layer is then mixed 1:1 ogous sources or stem cells for regenerative procedures. (v/v) with warm D-PBS/collagenase solution and subjected to Puregraft ® 250 System (Puregraft LLC, Solana Beach, CA), several cycles of centrifugation with serial aspiration of the and Lipogems (Lipogems, Norcross, GA) are specific Curr Rev Musculoskelet Med (2020) 13:264–280 269

Table 2 Enzymatic and mechanical methods of ADSC isolation

Product Company Design Publication/ patent

Mechanical Harvest® AdiPrep® Cha-Station™ Somnotec http://www.somnotec.net Syringe Emulsification and [10] Centrifugation Sieve Shaker PNC International Co., Ltd. http://inphronics.com. Semiclosed, semiautomated [70] sg/equipment/cha-station Octagon D200 Endecotts Ltd. https://www.endecotts.com Electromagnetic vibration machine [71] Lipokit with Medi-Khan http://www.medikanint.com/ Semiclosed, manual, centrifugation [70] Maxstem Puregraft 250 Puregraft LLC http://www.puregraft.com Semiclosed, manual, filtration [65] Lipogems Lipogems https://understandlipogems.com Closed, filtration [72] Enzymatic Rigenera cons HBW srl https://rigenerahbw.com/ Single-use, closed, centrifugation [60] Sepax™ C-Pro GE Healthcare https://www.gelifesciences.com Semiclosed, semiautomated [73] Celution® Cytori Therapeutics, Inc. http://www.cytori.com Semiclosed, automated, decantation, [70] 800/CRS +collagenase GID SVF-1 GID Group, Inc. http://www.thegidgroup.com/ Closed, manual, filtration, [74] +collagenase Tissue Genesis® Tissue Genesis, Inc. http://www.tissuegenesis.com/icellator Semiclosed, semiautomate, +adpiase [75] Icellator Cellthera Kit I and Cellthera, Ltd. https://www.cellthera.org Closed, automated, +collagenase [76] II

technologies designed to isolate ADSCs through non-invasive, in obtaining tissue. Bertozzi described a hybrid method for mechanical means. Studies have shown washing with filtration harvesting adipose tissues and isolating MSCs using a combi- in a closed system, such as Puregraft, produces a fat graft with nation of mechanical and enzymatic methods that produces higher tissue viability with few tissue contaminants. [65]Inbasic ready-to-use ADSCs pellet in 80 min. [81] science studies, Lipogem technology touts preserved exosome Another area that holds promise is augmentation of the activity given an intact stromal vascular niche, which increases AVF isolate with biologics. Sceldis® (ED Co. Ltd. & bioactivity in a paracrine fashion following MSC activation. The Purebiotech Co., Ltd., South Korea/Medica Group, United proposed benefit of this method is cartilage restoration that Arab Emirates) is one such technology that has been used in mimics adjacent tissue. [53, 72, 78, 79] orthopedics in which autologous SVF is injected along with Newer technologies are currently under development to pro- PRP, hyaluronic acid, and CaCl2 to treat knee pain following cure ADSCs in a mechanical fashion. One such tool, Rigenera meniscus tears. [82, 83] cons (HBW srl, Turin, Italy), is a disposable tool that decreases There is currently limited literature comparing the various the dimension of adipose tissue scraps in a non-enzymatic fash- technologies available. Moreover, there is a paucity of pro- ion and produces a cellular suspension injectable with a needle, spective studies evaluating the benefit of these preparations known as an adipose micro-graft. [80] The use of vibrational in isolated injections. Future research is warranted to better energy to isolate ADSCs has been described and theoretically clarify the components of these formulations and steps in may provide surgeons with another non-enzymatic method to preparation as to standardize this process. Standardization produce isolated ADSCs. The initial study on this technology and promulgation of these methods will allow for safer use demonstrated that ADSCs treated with a vibration machine, of ADSCs and for more homogenous comparisons of out- Sieve Shaker Ocatgon D200 (Endecotts Ltd., London, United comes between studies. Kingdom), did not affect the viability or proliferation of the cul- tured ADSCs. [71] However, the initial study failed to isolate ADSCs from IFP utilizing vibrational energy alone, suggesting Evidence from Animal Models that ADSCs from IFP may require alterations in the vibration frequencies utilized in the initial study. Therapeutic Mechanisms in Achilles Tendon Injury As various enzymatic and non-enzymatic technologies Models continue to be developed, the most successful technique will likely be the one that allows for the greatest amount of The use of ADSCs has been studied in various models of multipotent differentiation while ensuring ease and reliability induced injuries in animals. Lee and colleagues [84] obtained 270 Curr Rev Musculoskelet Med (2020) 13:264–280

ADSCs from the lipoaspirate of human subcutaneous fat tis- degradation gene expression, while increasing collagen syn- sue of healthy donors. They washed lipoaspirates with PBS thesis in comparison to controls. However, they showed that and digested them in PBS containing 1% bovine serum albu- the addition of ADSCs was more effective than CTGF alone min and 0.025% collagenase type I. Next, the isolated SVF in reducing inflammatory markers, increasing collagen, and was cultured to obtain a sufficient number of cells for injec- increasing tendon stem/progenitor cells at the tendon surface tion. Finally, ADSC were harvested by trypsinization, and along suture tracks. The authors proposed that this com- suspended, and tested for purity. Using these ADSCs, the bined approach using ADSCs may promote expedited flexor authors found that implantations of ADSCs in Sprague- tendon healing and warrants further investigation. [17] Dawley rats with full-thickness rectangular defects in the Achilles tendon led to better gross morphological and biome- Therapeutic Mechanisms in Rotator Cuff Injury chanical recovery than those in both the fibrin and sham Models groups. In addition, they found that ADSCs significantly in- creased the expression of human-specific type I collagen and Chen et al. [85] investigated the effects of ADSCs in a rat tenascin-1. The authors concluded that one of the benefits of model of collagenase-induced rotator cuff injury and com- ADSCs was the propensity to secrete proteins and provide pared their results to a control group that received a saline potential for therapeutic healing. placebo injection. In this study, ADSCs were provided by In a controlled laboratory study, Oshita et al. [14]investi- GWOXI Applied Technology Co., Ltd. (Hsinchu, Taiwan) gated the effects of ADSCs on Achilles tendon healing in 16 from a 3-g tissue sample from the infraumbilical region of a F344/NSIc rats that underwent collagenase injections in the 46-year-old Asian male. Processing was performed in a third- Achilles tendon to simulate tendinopathy. One week after this party laboratory where isolated ADSCs were cultured in injection, eight rats received ADSCs while eight received sa- Keratinocyte-SFM (Gibco, Grand Island, NY, USA), 10% line. ADSCs were harvested from the inguinal fat pads of two FBS (Hyclone, UT, USA), 50 μg/mL bovine pituitary extract F344/NSlc rats. Processing of ADSC consisted of finely minc- (Gibco), 5 ng/mL human recombinant epidermal growth fac- ing and enzymatically digesting the fat pads using 0.15% type tor (Gibco), 2 mM N-acetyl-l-cysteine (Sigma), and 0.2 mM l- I collagenase at 37 °C and adding an equal volume of DMEM ascorbic acid 2-phosphate (Sigma) under an atmosphere of containing 10% FBS for neutralization. At this point, the cells 5% CO2 at a temperature of 37 °C. The authors found that at were centrifuged at 1500 rpm for 5 min and seeded at a density 14 days post-ADSC injection, inflammatory cells were signif- of 105 cells/100-mm2 tissue culture plate and maintained in icantly decreased in this group compared to controls and that control medium of DMEM with 10% FBS and 1% antibiotic- the fiber arrangement and tendon organization were also su- antimycotic solution at 37 °C and 5% CO2.Finally,cellswere perior histologically. They also found that the load to failure of passaged using 0.25% trypsin when they reached 90% the ADSC group was significantly greater than in the control confluency. At both 4 and 12 weeks after treatment, the au- group (15.87 ± 2.20 N vs. 11.20 ± 1.35 N; p < 0.001), suggest- thors found that the ADSC group showed a significantly lower ing superior supraspinatus tensile strength conferred by the degree of tendon degeneration than the saline group. addition of ADSCs. Furthermore, on day 28, the injured ten- Furthermore, the type III to type I collagen ratio was signifi- don had a tensile strength equivalent to an uninjured tendon, cantly lower in the ADSCs group and this continued to de- suggesting full recovery. crease relative to the saline group. The authors concluded that The rotator cuff has also been the subject of several other ADSCs result in significant improvements in pathological studies. Notably, in one study concerning 50 Sprague-Dawley findings associated with tendinopathy (inflammation and rats that underwent detachment and repair of the supraspinatus micro-injury in tendons during the acute stage and a decrease tendon, animals were randomized to receive a collagen carrier in type I collagen in tendons). alone or collagen carrier with ADSCs. ADSCs were harvested from subcutaneous adipose tissue of two rats, after which the Therapeutic Mechanisms in Flexor Tendon Healing lipoaspirate was washed with PBS and digested at 37 °C for 30 min with collagenase. Ten percent FBS was used to neu- ADSCs have also been studied in controlled environments to tralize enzymatic activity, and the mixture was then centri- investigate their ability to aid tendon regeneration. The fol- fuged at 300g for 10 min. The resultant pellet was treated with lowing study did not report their method of ADSC harvesting 160 mM ammonium chloride for 10 min and then washed and and processing. Tendon regeneration in one study was stimu- suspended in DMEM plus 10% FBS. This was centrifuged lated using connective tissue growth factor (CTGF) with or again and the resulting cell suspension was filtered through a without ADSC. The ADSCs were applied to the repair surface 70-μm nylon mesh and the material obtained was resuspended of a canine flexor tendon using cell sheets, and the CTGF was in DMEM with glucose and pyruvate, 10% FBS, 2 mM glu- delivered via porous structures. At 14 days following repair, tamine, 1% streptomycin 10 μm/mL, and penicillin 1 UI/mL. both treatments reduced inflammatory markers and matrix The cells were then plated in 100-mm tissue-culture dishes at Curr Rev Musculoskelet Med (2020) 13:264–280 271

10 to 15 × 10 3 cells/mL and cultured at 37 °C in a humid Following 48 h of incubation at 37 °C/5%CO2, the cultures atmosphere with 5% carbon dioxide in DMEM containing were washed with PBS and maintained in stromal medium. 10% FBS and 1% penicillin/streptomycin. The medium was Non-adherent cells were aspirated with the medium after 24 h changed to remove non-adherent cells 24 h after seeding, and of cultivation and fresh medium was added and changed every every 4 days thereafter. After their experiment, the authors 3 days. The cells were sub-cultured three times by using stan- found no differences in biomechanical variables, but histolog- dard methods of trypsinization and cells achieved confluence ic examination demonstrated significantly less inflammation after 10 to 20 days when they were separated from the culture and edema in the ADSCs group. The authors proposed that flask by treating with 0.25% trypsin. The authors found that this may lead to a more elastic repair and less scarred healing. administration of these ADSCs delayed the progression of [16] osteoarthritic changes, including cartilage thinning, osteo- Similarly, Kim et al. [86] created bilateral supraspinatus phyte formation, and subchondral sclerosis in the joint. tears in 11 rabbits and repaired them after 3 weeks. They Ude et al. [89] administered intra-articular injections of randomized which supraspinatus received ADSCs at the time labeled autologous ADSCs into osteoarthritic sheep knees to of repair and injected the ADSCs at the muscle belly near the study their effect on cartilage regeneration. They harvested musculotendinous junction and saline in the control side. adipose tissue from the right infra patella fat pad of the sheep ADSCs were harvested from subcutaneous adipose tissue of during the surgical resections at week 1. Processing of the New Zealand male rabbits and digested with a type I collage- adipose consisted of mincing with a surgical blade before nase solution (Invitrogen, Carlsbad, CA, USA) with gentle digestion with an equal volume of 0.6% collagenase II agitation for 1 h at 37 °C. They separated the upper (Gibco) in an orbital incubator at 37 °C with 21-g force for fractions from stromal fractions by centrifugation at 1200g for 2 h. Subsequently, the digest was filtered with a cell strainer of 10 min. The remaining stromal fractions were treated with 100 μm pore size and centrifuged at a 4724-g force for 5 min 3-mL red blood cell lysis buffer (Sigma-Aldrich) for 10 min at 37 °C. The resultant pellet was washed with PBS twice and at room temperature, filtered through a 100-μm nylon mesh, basal medium before culture. The authors of this study found and centrifuged at 1200g for 10 min. The combined stromal that ADSCs preferentially populated to areas with osteoar- fractions of the samples were re-suspended and cultured in thritic change and decreased the progression of OA through- DMEM with 5% FBS and cells were allowed to adhere to out the knee. Furthermore, histological staining revealed the flask for 24 h at which point fresh media were added. loosely packed matrices of de novo cartilage, while immuno-

The cells were incubated at 37 °C and 5% CO2, and culture staining demonstrated the presence of the cartilage specific media were changed every 2–3 days. To perform the trans- proteins collagen II and SOX9. plantation, cells were suspended to a concentration of 1 × 107 Dasandro et al. [90] harvested adipose from the inguinal labeled cells/500 μLofHank’s balance salt solution (HBSS) zone of adult male New Zealand rabbits. This group processed and then were injected. The authors found significantly in- the adipose by treating it with 0.4 U/mL NB4 collagenase creased expression of both IFG-1 and myosin heavy chain standard grade, after which they re-suspended the SVF in α- (MyHC) in the ADSCs-injected rotator cuff, suggesting po- MEM (Gibco) supplemented with 1 U/mL heparin (Sigma, St tential anabolic effects of ADSCs use in rotator cuff Louis, MO, USA), 2% platelet growth factor enriched plasma pathology. (PGFEP), and 0.05 g/mL penicillin G (Gibco). After cultur- ing, cells were harvested and seeded at a density of 2000 cells/ Cartilage Regeneration in Animal Models cm2 for expansion. The selection of ADSCs was determined by ability to adhere to the plastic, to form colonies, and to ADSC therapy for treatment of osteoarthritis (OA) and differentiate into chondrogenic and osteogenic lineages. chondral defects in animal models has been well documented. After intra-articular administration into osteoarthritis-induced [87]Toghraieetal.[88] used ADSCs derived from knees of adult male New Zealand rabbits, the authors found infrapatellar fat pad (IFP) in rabbits subsequently administered that labeled ADSCs were detected in the synovial membrane to rabbits with OA-induced knees. They processed these and medial meniscus but not in the cartilage tissue at 3 to ADSCs as follows: the fat pad was minced and washed three 20 days after injection. At these same time periods, white to four times with PBS after which it was suspended in an cartilage with no degenerative noticeable macroscopic evi- equal volume of PBS with 1% FBS and 0.1% collagenase dence was observed in the ADSCs-treated groups. type I prewarmed to 37 °C. The tissue was subjected to con- Furthermore, a significant reduction of the fibrillin index and tinuous agitation for 3 h in a shaking bath and centrifuged for significant increase in cartilage thickness was found in the 5 min at 300g at room temperature. The supernatant was re- ADSC group compared to the control group. The decrease moved, and the pelleted stromal connective tissue fraction was in OA progression with ADSC suggests that there may be resuspended in culture medium (DMEM, 10% FBS, penicil- some trophic mechanism of action by release of growth fac- lin–streptomycin, actinomycine) and plated for culture. tors and cytokines that stimulate healing and chrondrogenesis. 272 Curr Rev Musculoskelet Med (2020) 13:264–280

These animal studies suggest that ADSC therapy may offer effective treatments for Achilles tendinopathy and that pa- patients who suffer from arthritis a non-invasive, safe, and tients with SVF may obtain faster results. easy option for treatment of arthritis. Together, the proposed evidence from the basic science Treatment of Rotator Cuff Tendinopathy literature confirms the propensity for ADSCs to differentiate into several biologic lineages which may serve therapeutic Clinical studies investigating the therapeutic effects of ADSCs functions in the realm of clinical sports medicine and preser- on rotator cuff tendinopathy are sparse. Striano et al. [94] vation efforts. Multiple studies within the past 5 years that injected micro-fragmented adipose tissue harvested from the have investigated the effects of ADSCs in animal models have abdomen and processed with Lipogems® (Lipogems USA, demonstrated the efficacy of this biologic and its potential for Atlanta Ga.) into 18 shoulders with varying cuff pathology. clinical use. [15, 85, 91, 92] Indeed, in many aspects of sports At 1-year follow-up, significant improvement in National Pain medicine where solutions for preservation are scarce, such as Scale (NPS) and American Shoulder and Elbow Surgeons cartilage regeneration and bone remodeling, ADSCs represent Score (ASES) were reported, from 7.5 to 3.6 and 33.7 to an area of potential to aid the controlled restoration of normal 69.2, respectively. Prospectively designed studies with large biological growth to promote healing. As discussed above, numbers of participants are required to further confirm the few studies currently exist which have investigated the use benefits of ADSCs in rotator cuff tendinopathy. of ADSCs as a source of mesenchymal stem cells in particular; however, the ease of harvesting human ADSCs in conjunction Treatment of Osteoarthritis with these early preliminary results suggest that future inves- tigations are worthwhile. Clinical studies involving ADSCs therapy suggest that these cells may offer a non-surgical treatment option for degenera- tive joint disease. A number of clinical studies have been Evidence from Clinical Studies conducted to understand the potential benefit of these inter- ventions in treating various degrees of osteoarthritis. The subjects of clinical studies which have investigated the Pers et al. [95] conducted a bicentric, uncontrolled phase I effects of ADSCs have primarily been restricted to the clinical trial in which 18 patients with severe osteoarthritis Achilles tendon, knee osteoarthritis, and osteochondral de- (OA) received a single intra-articular injection of autologous fects of the knee (Table 3). These studies represent heteroge- ADSC following. The study design consisted of a dose esca- neous populations in which many variations of ADSC formu- lation stratification. Three consecutive cohorts (six patients lations are employed. Despite these limitations, the current each) were created in which the first six received a low dose evidence suggests potential benefits for the use of ADSCs. (2 × 106 cells) injection of ADSCs, the next six received a medium dose (10 × 106), and the third six received a high dose Treatment of Achilles Tendinopathies (50 × 106). All patients underwent outpatient liposuction for adipose harvesting, and ADSCs were processed at a single Usuelli and colleagues [93]soughttocomparetheefficacyof facility (Etablissement Français du Sang Midi-Pyrénnées, PRP with the stromal vascular fraction of ADSCs (SVF) in 44 France) using a previously established and meticulous proto- patients with Achilles tendinopathy in a randomized, con- col. [107] At 6-month follow-up, the procedure was found to trolled trial (RCT). Adipose tissue was harvested from each be safe and associated with improved pain and function levels subject via lipoaspiration of abdominal subcutaneous tissue. It compared to baseline in all patients. [95] Panchal et al. [96] was also noted that two very thin patients were required to reported improvement in pain, quality of life, and functional have their adipose tissue harvested from the medial aspect of outcome scores at 12 months following ultrasound-guided their thighs. All adipose tissue was processed with the FastKit injection of micro-fractured adipose tissue into 25 arthritic system (Corios, San Giuliano Milanese, Italy) and mechani- knees (Kellgren-Lawrence, grade of 3 or 4). The average cally digested by rubbing tissue down through a 120-μmin- Knee Society Score (KSS) improved from 74 to 82, while ternal filter. The SVF was collected and centrifuged at 400g the lower extremity activity scale (LEAS) saw improvements for 10 min after which the resulting bottom phase was trans- from 36 to 47 at the 1-year post-injection timepoint. ferred to a new syringe and used for injections. Additional studies involving intra-articular injections of Postoperatively, the authors found a significant benefit for ADSCs into OA knees have reported associations with re- the SVF group compared to the PRP group when analyzing duced pain and improved knee function. [19, 20] the VISA-A, VAS pain, and AOFAS score at 15 and 30 days; Cattaneo et al. [97] sought to investigate the effects of however, this effect did not persist past this time point. Given autologous micro-fragmented adipose tissue injections in con- that all patients experienced improvement from baseline, the junction with arthroscopic procedures (meniscectomy or authors concluded that both PRP and SVF were safe and chondral shaving) for 38 patients with symptomatic knee urRvMsuokltMd(00 13:264 (2020) Med Musculoskelet Rev Curr Table 3 Evidence from human clinical studies for the use of ADSC

Study Subjects Location Comparison Results Conclusion design

Tendinopathy Usuellietal.[93] RCT 44 Achilles ADSC + PRP vs. PRP alone Early VAS pain, AOFAS, and PRP and SVF safe and effective; SVF may provide faster VISA-A score benefits results Striano et al. [94] Case Series 18 Rotator None Improvements in National Pain Scale Use of ADSCs resulted in significant improvements in Cuff and American Shoulder and Elbow pain, function and quality of life at 12 months Surgeons Score (ASES) Osteoarthritis

Pers et al. [95] Case Series 18 Knee None Improvements in KOOS, SAS, ADSCs are safe and well tolerated in patients with knee – 280 WOMAC VAS Pain osteoarthritis Panchal et al. [96] Case Series 17 Knee None Improvements in numerical pain The injection of autologous, micro-fractured, minimally rating scale, knee society score, and manipulated adipose tissueappearstobeasafeand lower extremity activity scale effective treatment option for patients with refractory, severe (grade 3 or 4) knee OA. Cattaneo et al. [97] Case Series 38 Knee None Improvements in KOOS and Micro-fragmented adipose tissue injection associated WOMAC scores with arthroscopic procedures is a safe and benefical adjunct for symptomatic knee OA Dall’Oca et al. [98] Case Series 6 Hip None Improvements in HHS and WOMAC MSC Lipogems is a fairly easy technique and no adverse scores effects were observed. Preliminary results showed a positive outcome. Hudetz et al. [99] Case Series 17 Knee None Increases in contents of cartilage The use of autologous and microfragmented adipose glycosaminoglycans; improved tissue in patients with knee OA (measured by VAS pain scores; no differences in dGEMRIC MRI) increased glycosaminoglycan (GAG) N-glycan or IgG synovial concen- content in hyaline cartilage, which is in line with ob- trations served clinical results Russo et al. [100] Case Series 22 Knee None Improvements in KOOS Lysholm, Autologous and micro-fragmented adipose tissue injec- VAS Pain, and IKDC scores tion is an innovative and safe approach for the man- agement of diffuse knee OA in the mid-term. Panni et al. [101] Case Series 52 Knee None Improvements in IKS and VAS pain Injection of ADSCs during arthroscopic debridement scores increased clinical and functional scores in patients with early knee OA at a mid-term follow-up, especially those with higher pre-operative VAS scores. Jo et al. 2014 [20] Case Series 18 Knee None Improvement in WOMAC score; size ADSC improved function and pain of the knee joint of cartilage defect decrease; volume without causing adverse events; reduced cartilage of cartilage increase; thick, defects hyaline-like cartilage regeneration. Koh et al. 2013 [19] Case Series 18 Knee None Improvement in WOMAC and ADSC effective and safe for improving function, Lysholm scores; decrease in VAS reducing pain pain; improved cartilage whole-organ MRI score Kim et al. 2015 [102] Cohort 20 Knee ADSC+PRP injection vs. ADSC Improved IKDC, Tegner scores implantation alone in implantation group 273 274 Table 3 (continued)

Study Subjects Location Comparison Results Conclusion design

ADSC implantation for knee OA resulted in better clinical and second-look arthroscopic outcomes than an ADSC injection. OCD Kim et al. [18] Case Series 55 Knee None Improved IKDC, Tegner scores Encouraging clinical outcomes Kim et al. [103] Cohort 49 Talus Marrow stimulation alone Improved VAS pain, AOFAS, and Encouraging results, even in setting of poor prognostic Tegner, cartilage restoration factors Kim et al. [104] Case Series 24 Knee None Improved IKDC, Tegner scores; ADSC useful for repairing cartilage lesions improved cartilage lesion grade on MRI Koh et al. [21] RCT 80 Knee Microfracture alone Improved KOOS, cartilage signal ADSC provided radiologic. KOOS pain and symptom intensity; 65% in ADSC group with subscore improvements; no differences in activity, complete cartilage coverage vs. sports, or quality-of-life 45% in control group Freitag et al. [105] Case Report 1 Knee None Improvement in NPRS pain score, The use of injection of autologous ADSCs following a Global WOMAC score, and all traumatic chondral defect of the patella resulted in components of the KOOS; MRI significant pain and functional improvements and showed complete filling of the complete regeneration of hyaline-like cartilage within chondral defect; modified ICRS the defect. score improved from Grade 3 to Grade 0. D’Ambrosi et al. Case Series 4 Talus None Improvements in the American Autologous microfractured, purified adipose tissue is safe [106] Orthopedic Foot and Ankle Society and effective for osteochondral lesions of the talus. score and VAS pain score; no complications. urRvMsuokltMd(00 13:264 (2020) Med Musculoskelet Rev Curr – 280 Curr Rev Musculoskelet Med (2020) 13:264–280 275

OA. Adipose tissue was obtained using a minimal manipula- 37.4 to 62.6 and the IKS function score improved from 57.2 to tion technique from the lower or lateral abdomen. The authors 83 at latest follow up. Kim et al. [102] in 2015 performed a processed all harvested adipose tissue using the Lipogems® match-paired cohort study of 40 patients in which one group processing kit. At a minimum 12-month follow-up, patients received arthroscopic debridement and injection of ADSCs on average demonstrated statistically significant improve- with PRP (n = 20), while another group underwent debride- ments from baseline in all components of the KOOS question- ment and implantation of ADSCs alone (n = 20). Adipose was naire and the WOMAC score. They also reported that all 38 harvested 1 day prior to surgery via tumescent liposuction of patients claimed to be satisfied with the treatment and each patient’s buttocks. Mature were processed that none were symptomatic, with the exception of one patient and separated from the SVF via a centrifugation protocol re- when performing squatting. No complications or adverse ported by Zuk et al. [43] and subsequently cultured. Using events were documented. epitope profiles, ADSCs were isolated. It was found that Dall’Oca et al. [98] retrospectively studied the outcomes of IKDC and Tegner scores had improved for both groups at six consecutive patients with hip OA who were treated with second look arthroscopy 1 year following the intervention. intra-articular injections of autologous ADSCs. Adipose tis- At 28.5 months, the mean IKDC and Tegner scores had im- sue was harvested from the abdominal wall and was processed proved to a significantly greater extent for the implantation using the Lipogems® processing kit. At 6-month follow-up, group when compared to the group who received an injection. patients demonstrated mean improvements in the Harris hip [102] score (67.2 vs. 84.6, p < 0.001), the WOMAC score (36.3 vs. 19.8, p < 0.001), and the VAS pain score (4.6 vs. 1.5, Treatment of Osteochondral Defects p < 0.001). One patient developed a hematoma on the abdo- men at the site of adipose tissue harvest. Several studies have focused solely on osteochondral defects Hudetz et al. [99] investigated the effects of intra-articular (OCDs) and attempted to elucidate the benefit of ADSCs injections of microfragmented adipose tissue on proteoglycan through patient reported outcomes, MRI findings, arthroscop- synthesis in patients with knee OA. The authors enrolled 17 ic findings, and histologic analysis. Kim et al. isolated and patients in a prospective, single-center, open-label clinical trial processed ADSCs as above and showed that mean pre- and and assessed through clinical and imaging outcomes as well as postoperative IKDC and Tegner activity scores significantly synovial fluid analysis at 12 months post-operatively. Adipose improved following arthroscopic debridement and ADSC in- harvest was procured from abdominal subcutaneous adipose jections in knees with isolated full-thickness cartilage lesions. tissue and was processed using the Lipogems® processing kit. Forty-one of 55 patients (74.5%) reported excellent/good re- At follow-up, the authors reported (1) decreases in VAS pain sults following surgery, with only 3 (5.5%) patients stating scores (3.9 vs. 0.6, p < 0.001), (2) no significant differences in they had poor results. [18] A study by the same author report- N-glycan or IgG synovial concentrations, and (3) increases in ed on functional outcomes at 2 years following arthroscopic cartilage glycosaminoglycans via dGEMRIC index values. marrow stimulation with injection of SVF containing ADSCs A recent study by Russo et al. [100] suggested that the versus marrow stimulation alone in osteochondral lesions of therapeutics effects of ADSCs for knee OA may persist as the talus. At mean follow-up of 21.9 months, all outcomes long as 3 years. Adipose tissue was harvested from the lateral including the VASpain, AOFAS, and Tegner scores improved or lower abdomen and was processed using the Lipogems® in the ADSC-intervention cohort compared to the marrow processing kit. They followed the outcomes of 30 patients, stimulation-only group. [103] The authors then divided pa- seven of which were excluded because they underwent addi- tients by median values for variables such as age, BMI, dura- tional procedures during the postoperative period (hyaluronic tion of symptoms, and lesion size to determine predictors of acid or platelet rich plasma injections), while another addition- outcomes. MRI follow-up scans showed more extensive car- al patient was lost to follow-up. In the remaining 22 patients, tilage restoration in the ADSC group, even in the setting of the authors reported median improvements in KOOS scores poor prognostic factors such as older age (> 46.1 years), large and Lysholm Knee scores of 20 points and 7 points, respec- lesion size (> 1.5 cm), and presence of subchondral cysts. tively. Furthermore, no adverse events were observed either at MRI follow-up studies have reported cartilage restoration the harvest sites or related to the knee joint. with ADSCs. Kim et al. isolated and processed ADSCs from Several studies have looked at injection of ADSCs follow- patients as reported in the previous studies by this group [18, ing arthroscopic debridement. Panni et al. [101] retrospective- 102, 103] and reported on isolated OCDs in 24 knees in which ly analyzed 52 patients with knee OA who received percuta- ADSC were implanted in a fibrin glue scaffold. Clinical out- neous injection of ADSC following arthroscopic debridement. comes measured with IKDC and Tegner activity scale scores Adipose tissue was harvested from the abdomen, and adipose improved and significantly correlated with similar improve- was processed using the Lipogems® processing kit. At 15- ments in cartilage lesion grades on MRI at 2-year follow-up. month follow up, the mean IKS knee score improved from [104] 276 Curr Rev Musculoskelet Med (2020) 13:264–280

Koh et al. [21] reported the results of a prospective RCT in significant improvements in the American Orthopedic Foot which patients with a symptomatic cartilage defect (> 3 cm2) and Ankle Society score (46.8 vs. 83.8, p < 0.05) and VAS were randomized to receive ADSCs with fibrin glue with pain score (8 vs. 2.3, p < 0.005), and no patients experienced microfracture treatment (n = 40) or microfracture alone (n = complications. 40) for the treatment of osteochondral lesions of the talus. This Together, the evidence from the few clinical studies avail- group harvested subcutaneous adipose tissue from the but- able shows promising outcomes in the treatment of select tocks of each patient 1 day prior to surgery and isolated the musculoskeletal pathologies. The limitation to most of this SVF and ADSC per the protocol outlined by Zuk et al. [43] published literature is the inclusion of other therapeutic bio- where following isolation ADSCs were characterized by ex- logics, such as PRP combined with ADSC, in the injection. pression of the surface markers CD90 and CD105 and absence Additionally, these studies lack a control group in most cases, of CD34 and CD14. At 2-year follow-up, 26 patients (65%) in making establishment of causation statistically inappropriate. the ADSC group had complete cartilage coverage of the lesion compared to 18 patients (45%) in the microfracture group alone. Better signal intensity was observed in the ADSC group Future Research as well. Additionally, KOOS pain and symptom sub-scores were improved. There was no reported difference in activity, The small body of literature that exists on ADSCs in human sports, and quality of life sub-scores. [21]Furthermore,the studies suggests that there is an associated benefit with injec- authors histologically examined microfracture-repaired tion of these cells into diseased joints or tissues. Many studies OCDs with and without ADSC and found that the mean have reported increased cartilage thickness and viability after International Cartilage Repair Society II score was 1054 for treatment with ADSCs. With regards to OA, the majority of ADSC group with microfracture compared to 967 for studies report clear and consistent benefits in terms of im- microfracture alone (p =0.036).[21] provements in function and pain when administering Freitag et al. [105] reported a case on the use of autologous ADSCs into the hip and knee joint. Similar benefits are dem- ADSCs for a post-traumatic chondral defect of the patella in a onstrated with studies concerning Achilles and rotator cuff 26-year-old female athlete. The patient initially underwent tendinopathies. However, the drawback on the design of these arthroscopic chondroplasty with chondral loose body removal studies is that most do not have a control group, and those that but failed to symptomatically improve 1 year post-operatively. do often still have some intervention in the control group, Repeat MRI at that point demonstrated a persistent patella whether that be PRP injection, microfracture, or some other chondral defect measuring 12 mm. At that point, the patient type of preservation procedure. Furthermore, these studies received intra-articular administration of ADSCs in conjunc- often use different protocols to extract and prepare their tion with arthroscopic microfracture to the area of full- ADSCs, and a variety of formulations have been found in thickness chondral loss. The source of adipose tissue was the the literature. The majority of these studies also use a case patient’s abdomen and ADSCs were isolated by separating the series design and cannot establish causation between ADSCs SVF through enzymatic digestion and centrifugation. The and these outcomes. As interest in the use of ADSCs con- SVF was subsequently cultured under hypoxic conditions in tinues to increase, it is imperative to both validate their safety 10% FBS, after which non-adherent cells were removed by and efficacy with higher quality, prospective studies using washing with PBS and expanded up to two passages. After consistent, predictable control populations, and to better stan- passage 2, cells were harvested, washed three times, and cryo- dardize formulations of ADSCs such that legitimate compar- preserved in clinical-grade cryoprotectant media using a vali- isons between studies can be made. The use of a standardized dated control rate freezing method of 1 °C/min. The patient tool to improve transparency and consistency of the process- experienced progressive improvement in pain (NPRS pain ing and harvesting of ADSCs, such as DOSES, [108] may be a score improved from 8 to 2 within 3 months), quality of life first step towards better understanding the characteristics and (Global WOMAC score improved from 64 to 92 at uses of ADSCs in clinical settings. 12 months), and all components of the KOOS. Furthermore, structural follow-up using MRI showed complete filling of the chondral defect and the modified ICRS score improved from Conclusion grade 3 to grade 0. D’Ambrosi et al. [106] used autologous microfractured and The proposed mechanisms of actions of ADSCs are in their purified adipose tissue to treat four patients with inherent capacity to differentiate into cartilage and bone and osteochondral lesions of the talus. Adipose tissue was harvest- to augment joint, muscle, and tendon preserving treatments. ed from the abdomen of each patient and processed using the Currently, there exist many different formulations and proto- Lipogems® processing kit. At 6 months postoperatively, the cols to prepare ADSCs, with little empirical evidence authors reported that all patients demonstrated statistically supporting or rejecting their use. The efficacy of ADSCs has Curr Rev Musculoskelet Med (2020) 13:264–280 277 been consistently demonstrated in animal models for the treat- cord blood, or adipose tissue. Stem Cells. 2006;24(5):1294–301. ment of many orthopedic pathologies, with few early human https://doi.org/10.1634/stemcells.2005-0342. 12. Peeters CM, Leijs MJ, Reijman M, van Osch GJ, Bos PK. Safety studies showing promising results. of intra-articular cell-therapy with culture-expanded stem cells in humans: a systematic literature review. Osteoarthr Cartil. Compliance with Ethical Standards 2013;21(10):1465–73. https://doi.org/10.1016/j.joca.2013.06. 025. 13. Zhu Y, Liu T, Song K, Fan X, Ma X, Cui Z. Adipose-derived stem Human and Animal Rights and Informed Consent This article does not cell: a better stem cell than BMSC. Cell Biochem Funct. contain any studies with human or animal subjects performed by any of 2008;26(6):664–75. https://doi.org/10.1002/cbf.1488. the authors. 14. Oshita T, Tobita M, Tajima S, Mizuno H. Adipose-derived stem cells improve collagenase-induced tendinopathy in a rat model. Am J Sports Med. 2016;44(8):1983–9. https://doi.org/10.1177/ 0363546516640750. References 15. Ricco S, Renzi S, Del Bue M, Conti V, Merli E, Ramoni R, et al. Allogeneic adipose tissue-derived mesenchymal stem cells in 1. Verhaegen F, Brys P, Debeer P. Rotator cuff healing after needling combination with platelet rich plasma are safe and effective in of a calcific deposit using platelet-rich plasma augmentation: a the therapy of superficial digital flexor tendonitis in the horse. randomized, prospective clinical trial. J Shoulder Elb Surg. Int J Immunopathol Pharmacol. 2013;26(1 Suppl):61–8. 2016;25(2):169–73. https://doi.org/10.1016/j.jse.2015.10.009. 16. Valencia Mora M, Antuna Antuna S, Garcia Arranz M, Carrascal 2. Ebert JR, Wang A, Smith A, Nairn R, Breidahl W, Zheng MH, MT, Barco R. Application of adipose tissue-derived stem cells in a et al. A midterm evaluation of postoperative platelet-rich plasma rat rotator cuff repair model. Injury. 2014;45(Suppl 4):S22–7. injections on arthroscopic supraspinatus repair: a randomized con- https://doi.org/10.1016/S0020-1383(14)70006-3. trolled trial. Am J Sports Med. 2017;45(13):2965–74. https://doi. 17. Shen H, Jayaram R, Yoneda S, Linderman SW, Sakiyama-Elbert org/10.1177/0363546517719048. SE, Xia Y, et al. The effect of adipose-derived stem cell sheets and 3. Flury M, Rickenbacher D, Schwyzer HK, Jung C, Schneider MM, CTGF on early flexor tendon healing in a canine model. Sci Rep. Stahnke K, et al. Does pure platelet-rich plasma affect postopera- 2018;8(1):11078. https://doi.org/10.1038/s41598-018-29474-8. tive clinical outcomes after arthroscopic rotator cuff repair? A 18. Kim YS, Choi YJ, Koh YG. Mesenchymal stem cell implantation randomized controlled trial. Am J Sports Med. 2016;44(8): in knee osteoarthritis: an assessment of the factors influencing 2136–46. https://doi.org/10.1177/0363546516645518. clinical outcomes. Am J Sports Med. 2015;43(9):2293–301. 4. de Windt TS, Vonk LA, Slaper-Cortenbach IC, van den Broek MP, https://doi.org/10.1177/0363546515588317. Nizak R, van Rijen MH, et al. Allogeneic mesenchymal stem cells 19. Koh YG, Jo SB, Kwon OR, Suh DS, Lee SW, Park SH, et al. stimulate cartilage regeneration and are safe for single-stage carti- Mesenchymal stem cell injections improve symptoms of knee – lage repair in humans upon mixture with recycled autologous osteoarthritis. Arthroscopy. 2013;29(4):748 55. https://doi.org/ chondrons. Stem Cells. 2017;35(1):256–64. https://doi.org/10. 10.1016/j.arthro.2012.11.017. 1002/stem.2475. 20. Jo CH, Lee YG, Shin WH, Kim H, Chai JW, Jeong EC, et al. Intra- articular injection of mesenchymal stem cells for the treatment of 5. Kaminski R, Kulinski K, Kozar-Kaminska K, Wielgus M, osteoarthritis of the knee: a proof-of-concept clinical trial. Stem Langner M, Wasko MK, et al. A prospective, randomized, dou- Cells. 2014;32(5):1254–66. https://doi.org/10.1002/stem.1634. ble-blind, parallel-group, placebo-controlled study evaluating meniscal healing, clinical outcomes, and safety in patients under- 21. Koh YG, Kwon OR, Kim YS, Choi YJ, Tak DH. Adipose-derived mesenchymal stem cells with microfracture versus microfracture going meniscal repair of unstable, complete vertical meniscal tears alone: 2-year follow-up of a prospective randomized trial. (bucket handle) augmented with platelet-rich plasma. Biomed Res – Int. 2018;2018:9315815. https://doi.org/10.1155/2018/9315815. Arthroscopy. 2016;32(1):97 109. https://doi.org/10.1016/j. arthro.2015.09.010. 6. Via AG, Frizziero A, Oliva F. Biological properties of mesenchy- 22. Caplan AI. Mesenchymal stem cells: time to change the name! mal stem cells from different sources. Muscles Ligaments Stem Cells Transl Med. 2017;6(6):1445–51. https://doi.org/10. Tendons J. 2012;2(3):154–62. 1002/sctm.17-0051. 7. Ruzzini L, Abbruzzese F, Rainer A, Longo UG, Trombetta M, 23. Gilboa L, Nohe A, Geissendorfer T, Sebald W, Henis YI, Knaus P. Maffulli N, et al. Characterization of age-related changes of ten- Bone morphogenetic protein receptor complexes on the surface of don stem cells from adult human tendons. Knee Surg Sports live cells: a new oligomerization mode for serine/threonine kinase – Traumatol Arthrosc. 2014;22(11):2856 66. https://doi.org/10. receptors. Mol Biol Cell. 2000;11(3):1023–35. https://doi.org/10. 1007/s00167-013-2457-4. 1091/mbc.11.3.1023. 8. De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, 24. Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from Zhu M, et al. Comparison of multi-lineage cells from human ad- cell membrane to nucleus through SMAD proteins. Nature. ipose tissue and bone marrow. Cells Tissues Organs. 2003;174(3): 1997;390(6659):465–71. https://doi.org/10.1038/37284. – 101 9. https://doi.org/10.1159/000071150. 25. Deregowski V,Gazzerro E, Priest L, Rydziel S, Canalis E. Notch 1 9. Dragoo JL, Samimi B, Zhu M, Hame SL, Thomas BJ, Lieberman overexpression inhibits osteoblastogenesis by suppressing Wnt/ JR, et al. Tissue-engineered cartilage and bone using stem cells beta-catenin but not bone morphogenetic protein signaling. J from human infrapatellar fat pads. J Bone Joint Surg (Br). Biol Chem. 2006;281(10):6203–10. https://doi.org/10.1074/jbc. 2003;85(5):740–7. M508370200. 10. Dragoo JL, Chang W. Arthroscopic harvest of adipose-derived 26. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. mesenchymal stem cells from the infrapatellar fat pad. Am J Osf2/Cbfa1: a transcriptional activator of differentia- Sports Med. 2017;45(13):3119–27. https://doi.org/10.1177/ tion. Cell. 1997;89(5):747–54. https://doi.org/10.1016/s0092- 0363546517719454. 8674(00)80257-3. 11. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative 27. Nobta M, Tsukazaki T, Shibata Y, Xin C, Moriishi T, Sakano S, analysis of mesenchymal stem cells from bone marrow, umbilical et al. Critical regulation of bone morphogenetic protein-induced 278 Curr Rev Musculoskelet Med (2020) 13:264–280

osteoblastic differentiation by Delta1/Jagged1-activated Notch1 method by human cartilage-derived progenitor cells: a comparison signaling. J Biol Chem. 2005;280(16):15842–8. https://doi.org/ with human adipose-derived mesenchymal stromal cells. Artif 10.1074/jbc.M412891200. Organs. 2013;37(12):1068–75. https://doi.org/10.1111/aor.12121. 28. Grottkau BE, Lin Y. Osteogenesis of adipose-derived stem cells. 43. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Bone Res. 2013;1(2):133–45. https://doi.org/10.4248/ Multilineage cells from human adipose tissue: implications for BR201302003. cell-based therapies. Tissue Eng. 2001;7(2):211–28. https://doi. 29. Rath B, Nam J, Knobloch TJ, Lannutti JJ, Agarwal S. org/10.1089/107632701300062859. Compressive forces induce osteogenic gene expression in 44. Merceron C, Vinatier C, Portron S, Masson M, Amiaud J, calvarial . J Biomech. 2008;41(5):1095–103. https:// Guigand L, et al. Differential effects of hypoxia on doi.org/10.1016/j.jbiomech.2007.11.024. osteochondrogenic potential of human adipose-derived stem cells. 30. Yang X, Gong P, Lin Y, Zhang L, Li X, Yuan Q, et al. Cyclic Am J Phys Cell Phys. 2010;298(2):C355–64. https://doi.org/10. tensile stretch modulates osteogenic differentiation of adipose- 1152/ajpcell.00398.2009. derived stem cells via the BMP-2 pathway. Arch Med Sci. 45. Gonzalez-Cruz RD, Darling EM. Adipose-derived stem cell fate is 2010;6(2):152–9. https://doi.org/10.5114/aoms.2010.13886. predicted by cellular mechanical properties. Adipocyte. 2013;2(2): 31. Arnsdorf EJ, Tummala P, Kwon RY, Jacobs CR. Mechanically 87–91. https://doi.org/10.4161/adip.23015. induced osteogenic differentiation–the role of RhoA, ROCKII 46. Xu J, Wang W, Ludeman M, Cheng K, Hayami T, Lotz JC, et al. and cytoskeletal dynamics. J Cell Sci. 2009;122(Pt 4):546–53. Chondrogenic differentiation of human mesenchymal stem cells https://doi.org/10.1242/jcs.036293. in three-dimensional alginate gels. Tissue Eng Part A. 2008;14(5): 32. Saler M, Caliogna L, Botta L, Benazzo F, Riva F, Gastaldi G. 667–80. https://doi.org/10.1089/tea.2007.0272. hASC and DFAT, multipotent stem cells for regenerative medi- 47. Xu Y, Balooch G, Chiou M, Bekerman E, Ritchie RO, Longaker cine: a comparison of their potential differentiation in vitro. Int J MT. Analysis of the material properties of early chondrogenic Mol Sci. 2017;18(12). https://doi.org/10.3390/ijms18122699. differentiated adipose-derived stromal cells (ASC) using an 33. D'Alimonte I, Mastrangelo F, Giuliani P, Pierdomenico L, in vitro three-dimensional micromass culture system. Biochem Marchisio M, Zuccarini M, et al. Osteogenic differentiation of Biophys Res Commun. 2007;359(2):311–6. https://doi.org/10. mesenchymal stromal cells: a comparative analysis between hu- 1016/j.bbrc.2007.05.098. man subcutaneous adipose tissue and dental pulp. Stem Cells Dev. 48. Steinert AF, Palmer GD, Pilapil C, Noth U, Evans CH, Ghivizzani 2017;26(11):843–55. https://doi.org/10.1089/scd.2016.0190. SC. Enhanced in vitro chondrogenesis of primary mesenchymal 34. Gu H, Huang Z, Yin X, Zhang J, Gong L, Chen J, et al. Role of c- stem cells by combined gene transfer. Tissue Eng Part A. Jun N-terminal kinase in the osteogenic and adipogenic differen- 2009;15(5):1127–39. https://doi.org/10.1089/ten.tea.2007.0252. tiation of human adipose-derived mesenchymal stem cells. Exp 49. Bianchi F, Maioli M, Leonardi E, Olivi E, Pasquinelli G, Valente Cell Res. 2015;339(1):112–21. https://doi.org/10.1016/j.yexcr. S, et al. A new nonenzymatic method and device to obtain a fat 2015.08.005. tissue derivative highly enriched in pericyte-like elements by mild 35. Lewallen EA, Jones DL, Dudakovic A, Thaler R, Paradise CR, mechanical forces from human lipoaspirates. Cell Transplant. Kremers HM, et al. Osteogenic potential of human adipose-tissue- 2013;22(11):2063– 77. https://doi.org/10.3727/ derived mesenchymal stromal cells cultured on 3D-printed porous 096368912X657855. structured titanium. Gene. 2016;581(2):95–106. https://doi.org/ 50. Bonaros N, Rauf R, Schachner T, Laufer G, Kocher A. Enhanced 10.1016/j.gene.2016.01.015. cell therapy for ischemic heart disease. Transplantation. 36. Brunner D, Frank J, Appl H, Schoffl H, Pfaller W, Gstraunthaler 2008;86(9):1151–60. https://doi.org/10.1097/TP. G. Serum-free cell culture: the serum-free media interactive online 0b013e3181880f9e. database. ALTEX. 2010;27(1):53–62. 51. Centeno CJ, Bashir J. Safety and regulatory issues regarding stem 37. Montoya F, Martinez F, Garcia-Robles M, Balmaceda-Aguilera C, cell therapies: one clinic's perspective. PM R. 2015;7(4 Suppl): Koch X, Rodriguez F, et al. Clinical and experimental approaches S4–7. https://doi.org/10.1016/j.pmrj.2014.12.013. to knee cartilage lesion repair and mesenchymal stem cell chon- 52. Maioli M, Rinaldi S, Santaniello S, Castagna A, Pigliaru G, drocyte differentiation. Biol Res. 2013;46(4):441–51. https://doi. Delitala A, et al. Radioelectric asymmetric conveyed fields and org/10.4067/S0716-97602013000400015. human adipose-derived stem cells obtained with a nonenzymatic 38. Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X, et al. method and device: a novel approach to multipotency. Cell Platelet lysates promote mesenchymal stem cell expansion: a safe- Transplant. 2014;23(12):1489–500. https://doi.org/10.3727/ ty substitute for animal serum in cell-based therapy applications. J 096368913X672037. Cell Physiol. 2005;205(2):228–36. https://doi.org/10.1002/jcp. 53. Striano RD, Chen H, Bilbool N, Azatullah K, Hilado J, Horan K. 20391. Case study: non-responsive knee pain with osteoarthritis and con- 39. Guilak F, Estes BT, Diekman BO, Moutos FT, Gimble JM. 2010 current meniscal disease treated with autologous micro- Nicolas Andry award: multipotent adult stem cells from adipose fragmented adipose tissue under continuous ultrasound guidance. tissue for musculoskeletal tissue engineering. Clin Orthop Relat CellR4. 2015;3(5):e1690. Res 2010;468(9):2530–2540. doi:https://doi.org/10.1007/s11999- 54. Thanunchai M, Hongeng S, Thitithanyanont A. Mesenchymal 010-1410-9. stromal cells and viral infection. Stem Cells Int. 2015;2015: 40. Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, 860950. https://doi.org/10.1155/2015/860950. Pittenger MF. Chondrogenic differentiation of cultured human 55. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, mesenchymal stem cells from marrow. Tissue Eng. 1998;4(4): Krause U, et al. Comparative characteristics of mesenchymal stem 415–28. https://doi.org/10.1089/ten.1998.4.415. cells from human bone marrow, adipose tissue, and umbilical cord 41. Felimban R, Ye K, Traianedes K, Di Bella C, Crook J, Wallace blood. Exp Hematol. 2005;33(11):1402–16. https://doi.org/10. GG, et al. Differentiation of stem cells from human infrapatellar 1016/j.exphem.2005.07.003. fat pad: characterization of cells undergoing chondrogenesis. 56. Caplan AI. Adult mesenchymal stem cells for tissue engineering Tissue Eng A. 2014;20(15–16):2213–23. https://doi.org/10.1089/ versus regenerative medicine. J Cell Physiol. 2007;213(2):341–7. ten.tea.2013.0657. https://doi.org/10.1002/jcp.21200. 42. Baptista LS, Silva KR, Pedrosa CS, Amaral RJ, Belizario JV, 57. Vallee M, Cote JF, Fradette J. Adipose-tissue engineering: taking Borojevic R, et al. Bioengineered cartilage in a scaffold-free advantage of the properties of human adipose-derived stem/ Curr Rev Musculoskelet Med (2020) 13:264–280 279

stromal cells. Pathol Biol (Paris). 2009;57(4):309–17. https://doi. technology. Tissue Eng C Methods. 2012;18(8):575–82. https:// org/10.1016/j.patbio.2008.04.010. doi.org/10.1089/ten.TEC.2011.0617. 58. Dragoo JL, Lieberman JR, Lee RS, Deugarte DA, Lee Y, Zuk PA, 74. Dos-Anjos Vilaboa S, Navarro-Palou M, Llull R. Age influence et al. Tissue-engineered bone from BMP-2-transduced stem cells on stromal vascular fraction cell yield obtained from human derived from human fat. Plast Reconstr Surg. 2005;115(6):1665– lipoaspirates. Cytotherapy. 2014;16(8):1092–7. https://doi.org/ 73. https://doi.org/10.1097/01.prs.0000161459.90856.ab. 10.1016/j.jcyt.2014.02.007. 59. Estes BT, Diekman BO, Gimble JM, Guilak F. Isolation of 75. Doi K, Tanaka S, Iida H, Eto H, Kato H, Aoi N, et al. Stromal adipose-derived stem cells and their induction to a chondrogenic vascular fraction isolated from lipo-aspirates using an automated phenotype. Nat Protoc. 2010;5(7):1294–311. https://doi.org/10. processing system: bench and bed analysis. J Tissue Eng Regen 1038/nprot.2010.81. Med. 2013;7(11):864–70. https://doi.org/10.1002/term.1478. 60. Coleman SR. Structural fat grafting: more than a permanent filler. 76. Michalek J, Vrablikova A, Darinskas A, Lukac L, Prucha J, Plast Reconstr Surg. 2006;118(3 Suppl):108S–20S. https://doi. Skopalik J, et al. Stromal vascular fraction cell therapy for osteo- org/10.1097/01.prs.0000234610.81672.e7. arthritis in elderly: multicenter case-control study. J Clin Orthop 61. Klein JA. Tumescent technique for local anesthesia improves safe- Trauma. 2019;10(1):76–80. https://doi.org/10.1016/j.jcot.2018. ty in large-volume liposuction. Plast Reconstr Surg. 1993;92(6): 11.010. – 1085 98 discussion 99-100. 77. Alexander RW, Harrell DB. Autologous fat grafting: use of closed 62. Illouz YG. Refinements in the lipoplasty technique. Clin Plast syringe microcannula system for enhanced autologous structural – Surg. 1989;16(2):217 33. grafting. Clin Cosmet Investig Dermatol. 2013;6:91–102. https:// 63. Carvalho PP, Gimble JM, Dias IR, Gomes ME, Reis RL. Xenofree doi.org/10.2147/CCID.S40575. enzymatic products for the isolation of human adipose-derived 78. Ding X, Zhu M, Xu B, Zhang J, Zhao Y, Ji S, et al. Integrated – stromal/stem cells. Tissue Eng Part C Methods. 2013;19(6):473 trilayered silk fibroin scaffold for osteochondral differentiation of 8. https://doi.org/10.1089/ten.TEC.2012.0465. adipose-derived stem cells. ACS Appl Mater Interfaces. 64. Domenis R, Lazzaro L, Calabrese S, Mangoni D, Gallelli A, 2014;6(19):16696–705. https://doi.org/10.1021/am5036708. Bourkoula E, et al. Adipose tissue derived stem cells: in vitro 79. Carelli S, Messaggio F, Canazza A, Hebda DM, Caremoli F, and in vivo analysis of a standard and three commercially avail- Latorre E, et al. Characteristics and properties of mesenchymal able cell-assisted lipotransfer techniques. Stem Cell Res Ther. stem cells derived from microfragmented adipose tissue. Cell 2015;6:2. https://doi.org/10.1186/scrt536. Transplant. 2015;24(7):1233–52. https://doi.org/10.3727/ 65. Zhu M, Cohen SR, Hicok KC, Shanahan RK, Strem BM, Yu JC, 096368914X681603. et al. Comparison of three different fat graft preparation methods: 80. De Francesco F, Mannucci S, Conti G, Dai Pre E, Sbarbati A, gravity separation, centrifugation, and simultaneous washing with Riccio M. A non-enzymatic method to obtain a fat tissue deriva- filtration in a closed system. Plast Reconstr Surg. 2013;131(4): tive highly enriched in adipose stem cells (ASCs) from human 873–80. https://doi.org/10.1097/PRS.0b013e31828276e9. lipoaspirates: preliminary results. Int J Mol Sci. 2018;19(7). 66. Patrikoski M, Juntunen M, Boucher S, Campbell A, Vemuri MC, https://doi.org/10.3390/ijms19072061. Mannerstrom B, et al. Development of fully defined xeno-free 81. Raposio E, Bertozzi N. How to isolate a ready-to-use adipose- culture system for the preparation and propagation of cell derived stem cells pellet for clinical application. Eur Rev Med therapy-compliant human adipose stem cells. Stem Cell Res Pharmacol Sci. 2017;21(18):4252–60. Ther. 2013;4(2):27. https://doi.org/10.1186/scrt175. 67. Aguena M, Fanganiello RD, Tissiani LA, Ishiy FA, Atique R, 82. Pak J, Lee JH, Lee SH. Regenerative repair of damaged meniscus Alonso N, et al. Optimization of parameters for a more efficient with autologous adipose tissue-derived stem cells. Biomed Res use of adipose-derived stem cells in regenerative medicine thera- Int. 2014;2014:436029. https://doi.org/10.1155/2014/436029. pies. Stem Cells Int. 2012;2012:303610. https://doi.org/10.1155/ 83. Pak J, Chang JJ, Lee JH, Lee SH. Safety reporting on implantation 2012/303610. of autologous adipose tissue-derived stem cells with platelet-rich 68. Yang XF, He X, He J, Zhang LH, Su XJ, Dong ZY, et al. High plasma into human articular joints. BMC Musculoskelet Disord. efficient isolation and systematic identification of human adipose- 2013;14:337. https://doi.org/10.1186/1471-2474-14-337. derived mesenchymal stem cells. J Biomed Sci. 2011;18:59. 84. Lee SY, Kwon B, Lee K, Son YH, Chung SG. Therapeutic mech- https://doi.org/10.1186/1423-0127-18-59. anisms of human adipose-derived mesenchymal stem cells in a rat – 69. Kirkpatrick CJ, Melzner I, Goller T. Comparative effects of tryp- tendon injury model. Am J Sports Med. 2017;45(6):1429 39. sin, collagenase and mechanical harvesting on cell membrane https://doi.org/10.1177/0363546517689874. lipids studied in monolayer-cultured endothelial cells and a green 85. Chen HS, Su YT, Chan TM, Su YJ, Syu WS, Harn HJ, et al. monkey kidney cell line. Biochim Biophys Acta. 1985;846(1): Human adipose-derived stem cells accelerate the restoration of 120–6. https://doi.org/10.1016/0167-4889(85)90117-x. tensile strength of tendon and alleviate the progression of rotator 70. Aronowitz JA, Ellenhorn JD. Adipose stromal vascular fraction cuff injury in a rat model. Cell Transplant. 2015;24(3):509–20. isolation: a head-to-head comparison of four commercial cell sep- https://doi.org/10.3727/096368915X686968. aration systems. Plast Reconstr Surg. 2013;132(6):932e–9e. 86. Kim SH, Chung SW, Oh JH. Expression of insulin-like growth https://doi.org/10.1097/PRS.0b013e3182a80652. factor type 1 receptor and myosin heavy chain in rabbit's rotator 71. Packer JD, Chang WT, Dragoo JL. The use of vibrational energy cuff muscle after injection of adipose-derived stem cell. Knee Surg to isolate adipose-derived stem cells. Plast Reconstr Surg Glob Sports Traumatol Arthrosc. 2014;22(11):2867–73. https://doi.org/ Open. 2018;6(1):e1620. https://doi.org/10.1097/GOX. 10.1007/s00167-013-2560-6. 0000000000001620. 87. Nazempour A, Van Wie BJ. Chondrocytes, mesenchymal stem 72. Tremolada C, Colombo V, Ventura C. Adipose tissue and cells, and their combination in articular cartilage regenerative Mesenchymal stem cells: state of the art and Lipogems(R) tech- medicine. Ann Biomed Eng. 2016;44(5):1325–54. https://doi. nology development. Curr Stem Cell Rep. 2016;2:304–12. https:// org/10.1007/s10439-016-1575-9. doi.org/10.1007/s40778-016-0053-5. 88. Toghraie FS, Chenari N, Gholipour MA, Faghih Z, Torabinejad S, 73. Guven S, Karagianni M, Schwalbe M, Schreiner S, Farhadi J, Dehghani S, et al. Treatment of osteoarthritis with infrapatellar fat Bula S, et al. Validation of an automated procedure to isolate pad derived mesenchymal stem cells in rabbit. Knee. 2011;18(2): human adipose tissue-derived cells by using the Sepax(R) 71–5. https://doi.org/10.1016/j.knee.2010.03.001. 280 Curr Rev Musculoskelet Med (2020) 13:264–280

89. Ude CC, Sulaiman SB, Min-Hwei N, Hui-Cheng C, Ahmad J, fat tissue on proteoglycan synthesis in patients with knee osteoar- Yahaya NM, et al. Cartilage regeneration by chondrogenic in- thritis. Genes (Basel). 2017;8(10). https://doi.org/10.3390/ duced adult stem cells in osteoarthritic sheep model. PLoS One. genes8100270. 2014;9(6):e98770. https://doi.org/10.1371/journal.pone.0098770. 100. Russo A, Screpis D, Di Donato SL, Bonetti S, Piovan G, Zorzi C. 90. Desando G, Cavallo C, Sartoni F, Martini L, Parrilli A, Veronesi F, Autologous micro-fragmented adipose tissue for the treatment of et al. Intra-articular delivery of adipose derived stromal cells at- diffuse degenerative knee osteoarthritis: an update at 3 year fol- tenuates osteoarthritis progression in an experimental rabbit mod- low-up. J Exp Orthop. 2018;5(1):52. https://doi.org/10.1186/ el. Arthritis Res Ther. 2013;15(1):R22. https://doi.org/10.1186/ s40634-018-0169-x. ar4156. 101. Schiavone Panni A, Vasso M, Braile A, Toro G, De Cicco A, 91. Geburek F, Roggel F, van Schie HTM, Beineke A, Estrada R, Viggiano D, et al. Preliminary results of autologous adipose- Weber K, et al. Effect of single intralesional treatment of surgically derived stem cells in early knee osteoarthritis: identification of a induced equine superficial digital flexor tendon core lesions with subpopulation with greater response. Int Orthop. 2019;43(1):7– adipose-derived mesenchymal stromal cells: a controlled experi- 13. https://doi.org/10.1007/s00264-018-4182-6. mental trial. Stem Cell Res Ther. 2017;8(1):129. https://doi.org/ 102. Kim YS, Kwon OR, Choi YJ, Suh DS, Heo DB, Koh YG. 10.1186/s13287-017-0564-8. Comparative matched-pair analysis of the injection versus implan- 92. Canapp SO Jr, Canapp DA, Ibrahim V,Carr BJ, Cox C, Barrett JG. tation of mesenchymal stem cells for knee osteoarthritis. Am J The use of adipose-derived progenitor cells and platelet-rich plas- Sports Med. 2015;43(11):2738–46. https://doi.org/10.1177/ ma combination for the treatment of supraspinatus tendinopathy in 0363546515599632. 55 dogs: a retrospective study. Front Vet Sci. 2016;3:61. https:// 103. Kim YS, Lee HJ, Choi YJ, Kim YI, Koh YG. Does an injection of doi.org/10.3389/fvets.2016.00061. a stromal vascular fraction containing adipose-derived mesenchy- 93. Usuelli FG, Grassi M, Maccario C, Vigano M, Lanfranchi L, mal stem cells influence the outcomes of marrow stimulation in Alfieri Montrasio U, et al. Intratendinous adipose-derived stromal osteochondral lesions of the talus? A clinical and magnetic reso- vascular fraction (SVF) injection provides a safe, efficacious treat- nance imaging study. Am J Sports Med. 2014;42(10):2424–34. ment for Achilles tendinopathy: results of a randomized controlled https://doi.org/10.1177/0363546514541778. clinical trial at a 6-month follow-up. Knee Surg Sports Traumatol 104. Kim YS, Choi YJ, Lee SW, Kwon OR, Suh DS, Heo DB, et al. Arthrosc. 2018;26(7):2000–10. https://doi.org/10.1007/s00167- Assessment of clinical and MRI outcomes after mesenchymal 017-4479-9. stem cell implantation in patients with knee osteoarthritis: a pro- 94. Striano RD, Malanga GA, Bilbool N, Khatira A. Refractory shoul- spective study. Osteoarthr Cartil. 2016;24(2):237–45. https://doi. der pain with osteoarthritis, and rotator cuff tear, Treated With org/10.1016/j.joca.2015.08.009. Micro Fragmented Adipose Tissue. J Orthopaedics Spine Sports Med. 2018;1:1–5. 105. Freitag J, Li D, Wickham J, Shah K, Tenen A. Effect of autologous 95. Pers YM, Rackwitz L, Ferreira R, Pullig O, Delfour C, Barry F, adipose-derived mesenchymal stem cell therapy in the treatment et al. Adipose mesenchymal stromal cell-based therapy for severe of a post-traumatic chondral defect of the knee. BMJ Case Rep. osteoarthritis of the knee: a phase I dose-escalation trial. Stem 2017;2017. https://doi.org/10.1136/bcr-2017-220852. Cells Transl Med. 2016;5(7):847–56. https://doi.org/10.5966/ 106. D'Ambrosi R, Indino C, Maccario C, Manzi L, Usuelli FG. sctm.2015-0245. Autologous microfractured and purified adipose tissue for arthro- 96. Panchal J, Malanga G, Sheinkop M. Safety and efficacy of percu- scopic management of osteochondral lesions of the talus. J Vis taneous injection of lipogems micro-fractured adipose tissue for Exp. 2018;131. https://doi.org/10.3791/56395. osteoarthritic knees. Am J Orthop (Belle Mead NJ). 2018;47(11). 107. Bura A, Planat-Benard V,Bourin P, Silvestre JS, Gross F, Grolleau https://doi.org/10.12788/ajo.2018.0098. JL, et al. Phase I trial: the use of autologous cultured adipose- 97. Cattaneo G, De Caro A, Napoli F, Chiapale D, Trada P, Camera A. derived stroma/stem cells to treat patients with non- Micro-fragmented adipose tissue injection associated with arthro- revascularizable critical limb ischemia. Cytotherapy. 2014;16(2): scopic procedures in patients with symptomatic knee osteoarthri- 245–57. https://doi.org/10.1016/j.jcyt.2013.11.011. tis. BMC Musculoskelet Disord. 2018;19(1):176. https://doi.org/ 108. Murray IR, Chahla J, Safran MR, Krych AJ, Saris DBF, Caplan 10.1186/s12891-018-2105-8. AI, et al. International expert consensus on a cell therapy commu- 98. Dall'Oca C, Breda S, Elena N, Valentini R, Samaila EM, Magnan nication tool: DOSES. J Bone Joint Surg Am. 2019;101(10):904– B. Mesenchymal Stem Cells injection in hip osteoarthritis: prelim- 11. https://doi.org/10.2106/JBJS.18.00915. inary results. Acta Biomed. 2019;90(1-S):75–80. https://doi.org/ 10.23750/abm.v90i1-S.8084. Publisher’sNoteSpringer Nature remains neutral with regard to jurisdic- 99. Hudetz D, Boric I, Rod E, Jelec Z, Radic A, Vrdoljak T, et al. The tional claims in published maps and institutional affiliations. effect of intra-articular injection of autologous microfragmented